The basic immunoglobulin (Ig) structural unit in vertebrate systems is composed of two identical "light" polypeptide chains (approximately 23 kDa), and two identical "heavy" chains (approximately 53 to 70 kDa). The four chains are joined by disulfide bonds in a "Y" configuration, and the "tail" portions of the two heavy chains are bound by covalent disulfide linkages when the immunoglobulins are generated either by B cell hybridomas or other cell types.
A schematic of the general antibody structure is shown in FIG. 1. The light and heavy chains are each composed of a variable region at the N-terminal end, and a constant region at the C-terminal end. In the light chain, the variable region (termed "V.sub.L J.sub.L ") is composed of a variable (V.sub.L) region connected through the joining (J.sub.L) region to the constant region (C.sub.L). In the heavy chain, the variable region (V.sub.H D.sub.H J.sub.H) is composed of a variable (V.sub.H) region linked through a combination of the diversity (D.sub.H) region and the joining (J.sub.H) region to the constant region (C.sub.H). The V.sub.L J.sub.L and V.sub.H D.sub.H J.sub.H regions of the light and heavy chains, respectively, are associated at the tips of the Y to form the antibody's antigen binding portion and determine antigen binding specificity.
The (C.sub.H) region defines the antibody's isotype, i.e., its class or subclass. Antibodies of different isotypes differ significantly in their effector functions, such as the ability to activate complement, bind to specific receptors (e.g., Fc receptors) present on a wide variety of cell types, cross mucosal and placental barriers, and form polymers of the basic four-chain IgG molecule.
Antibodies are categorized into "classes" according to the C.sub.H type utilized in the immunoglobulin molecule (IgM, IgG, IgD, IgE, or IgA). There are at least five types of C.sub.H genes (C.mu., C.gamma., C.delta., C.epsilon., and C.alpha.), and some species (including humans) have multiple C.sub.H subtypes (e.g., C.gamma..sub.1, C.gamma..sub.2, C.gamma..sub.3, and C.gamma..sub.4 in humans). There are a total of nine C.sub.H genes in the haploid genome of humans, eight in mouse and rat, and several fewer in many other species. In contrast, there are normally only two types of light chain constant regions (C.sub.L) , kappa (.kappa.) and lambda (.lambda.), and only one of these constant regions is present in a single light chain protein (i.e., there is only one possible light chain constant region for every V.sub.L J.sub.L produced). Each heavy chain class can be associated with either of the light chain classes (e.g., a C.sub.H .gamma. region can be present in the same antibody as either a .kappa. or .lambda. light chain), although the constant regions of the heavy and light chains within a particular class do not vary with antigen specificity (e.g., an IgG antibody always has a C.gamma. heavy chain constant region regardless of the antibody's antigen specificity).
Each of the V, D, J, and C regions of the heavy and light chains are encoded by distinct genomic sequences. Antibody diversity is generated by recombination between the different V.sub.H, D.sub.H, and J.sub.H gene segments in the heavy chain, and V.sub.L and J.sub.L gene segments in the light chain. The recombination of the different V.sub.H, D.sub.H, and J.sub.H genes is accomplished by DNA recombination during B cell differentiation. Briefly, the heavy chain sequence recombines first to generate a D.sub.H J.sub.H complex, and then a second recombinatorial event produces a V.sub.H D.sub.H J.sub.H complex. A functional heavy chain is produced upon transcription followed by splicing of the RNA transcript. Production of a functional heavy chain triggers recombination in the light chain sequences to produce a rearranged V.sub.L J.sub.L region which in turn forms a functional V.sub.L J.sub.L C.sub.L region, i.e., the functional light chain.
During the course of B cell differentiation, progeny of a single B cell can switch the expressed immunoglobulin isotype from IgM to IgG or other classes of immunoglobulin without changing the antigen specificity determined by the variable region. This phenomenon, known as immunoglobulin class-switching, is accompanied by DNA rearrangement that takes place between switch (S) regions located 5' to each C.sub.H gene (except for C.gamma.) (reviewed in Honjo (1983) Annu. Rev. Immunol. 1:499-528, and Shimizu & Honjo (1984) Cell 36:801-803). S--S recombination brings the V.sub.H D.sub.H J.sub.H exon to the proximity of the C.sub.H gene to be expressed by deletion of intervening C.sub.H genes located on the same chromosome. The class-switching mechanism is directed by cytokines (Mills et al. (1995) J. Immunol. 155:3021-3036). Switch regions vary in size from 1 kb (S.epsilon.) to 10 kb (S.gamma..sub.1), and are composed of tandem repeats that vary both in length and sequence (Gritzmacher (1989) Crit. Rev. Immunol. 9:173-200). Several switch regions have been characterized including the murine S.mu., S.epsilon., S.alpha., S.gamma.3, S.gamma.1, S.gamma.2b and S.gamma.2a switch regions and the human S.mu. switch region (Mills et al. (1995) supra; Nikaido et al. (1981) Nature 292:845-8; Marcu et al. (1982) Nature 298:87-89; Takahashi et al. (1982) Cell 29:671-9; Mills et al. (1990) Nucleic Acids Res. 18:7305-16; Nikaido et al. (1982) J. Biol. Chem. 257:7322-29; Stanton et al. (1982) Nucleic Acids Res. 10:5993-6006; Gritzmacher (1989) supra; Davis et al. (1980) Science 209:1360; Obata et al. (1981) Proc. Natl. Acad. Sci. U.S.A. 78:2437-41; Kataoka et al. (1981) Cell 23:357; Mowatt et al. (1986) J. Immunol. 136:2674-83; Szurek et al. (1985) J. Immunol. 135:620-6; and Wu et al. (1984) EMBO J. 3:2033-40).
Observations that a single B cell can express more than one isotype simultaneously on its surface is not explained by the class-switching mechanism since S--S recombination is limited to intrachromosomal recombination and results in deletion of the exchanged C.sub.H gene. A second mechanism, called trans-splicing, has been described in which two transcripts generated from different chromosomes are joined to form a single continuous transcript (Shimizu et al. (1991) J. Exp. Med. 173:1385-1393). Transgenic mice carrying a rearranged expressible V.sub.H D.sub.H J.sub.H heavy chain .mu. gene integrated outside the mouse IgH locus were found to produce mRNA having the V.sub.H D.sub.H J.sub.H region of the transgene correctly spliced to the endogenous C.sub.H region. As with S--S recombination, the frequency of trans-splicing is low, and the factors regulating both mechanisms are not well understood.
The value and potential of antibodies as diagnostic and therapeutic reagents has been long-recognized in the art. Unfortunately, the field has been hampered by the slow, tedious processes required to produce large quantities of an antibody of a desired specificity. The classical cell fusion techniques allowed for efficient production of monoclonal antibodies by fusing the B cell producing the antibody with an immortalized cell line. The resulting cell line is called a hybridoma cell line. However, most of these monoclonal antibodies are produced in murine systems and are recognized as "foreign" proteins by the human immune system. Thus the patient's immune system elicits a response against the antibodies, which results in antibody neutralization and clearance, and/or potentially serious side-effects associated with the anti-antibody immune response.
One approach to this problem has been to develop human or "humanized" monoclonal antibodies, which are not as easily "recognized" as foreign epitopes, and avoid an anti-antibody immune response in the patient. Applications of human B cell hybridoma-produced monoclonal antibodies have promising potential in the treatment of cancer, microbial, and viral infections, B cell immunodeficiencies associated with abnormally low antibody production, autoimmune diseases, inflammation, transplant rejection and other disorders of the immune system, and other diseases. However, several obstacles remain in the development of such human monoclonal antibodies. For example, many human tumor antigens may not be immunogenic in humans and thus it may be difficult to isolate human B cells producing antibodies against human antigens.
Attempts to address the problems associated with antibodies for human therapeutics have used recombinant DNA techniques. Most of these efforts have focused on the production of chimeric antibodies having a human C.sub.H region and non-human (e.g., murine) antigen combining (variable) regions. These chimeric antibodies are generally produced by cloning the desired antibody variable region and/or constant region, combining the cloned sequences into a single construct encoding all or a portion of a functional chimeric antibody having the desired variable and constant regions, introducing the construct into a cell capable of expressing antibodies, and selecting cells that stably express the chimeric antibody. Alternatively, the chimeric antibody is produced by cloning the desired variable region or constant region, introducing the construct into an antibody-producing cell, and selecting for chimeric antibody-producing cells that result from homologous recombination between the desired variable region and the endogenous variable region, or the desired constant region and the endogenous constant region. Examples of techniques which rely upon recombinant DNA techniques such as those described above to produce chimeric antibodies are described in PCT Publication No. WO 86/01533 (Neuberger et al.), and in U.S. Pat. Nos. 4,816,567 (Cabilly et al.) and 5,202,238 (Fell et al.). These methods require transferring DNA from one cell to another, thus removing it from its natural locus, and thus require careful in vitro manipulation of the DNA to ensure that the final antibody-encoding construct is functional (e.g., is capable of transcription and translation of the desired gene product).
There is a clear need in the field for a method for producing a desired protein or antibody which does not require multiple cloning steps, in more efficient than conventional homologous recombination, and can be carried out in hybridoma cells.